The present Program Project focuses on three major aspects of PrPsc: 1) mode of formation; 2) physical and chemical characteristics; 3) mechanisms of pathogenicity. Four Research Projects are proposed. Project by Surewicz addresses the critical issue of PrPsc conformation using the PrPsc-like recombinant PrP developed in our group. Mechanisms of amyloid fiber formation by the Yl45Stop mutant PrP, which generates an N-terminal PrP fragment, as well as the effect of other mutations on the conformation of PrPsc will also be examined. Project by Chen proposes a comparative study of PrPsc in human and animal prion diseases. Characteristics that will be analyzed include gel migration and pattern of glycosylation. In addition, N-terminal protease cleavage sites, beta-sheet structure and aggregation of distinct PrPsc species will be determined and correlated. Project by Gambetti deals with the regions of human PrPsc that carry the infectivity and with the role of glycans in PrPsc strain formation as well as disease phenotype determination. Furthermore, the mechanisms of PrP to PrPsc conversion likely to vary in different mutations will be examined in cell models of genetic prion diseases. Project by Singh iis dedicated to studying the important issue of intercellular spreading of PrPsc. It will be first assessed whether synthetic and naturally occurring short PrP peptides are transported through a monolayer of intestinal and endothelial cells. Then, similar studies will be carried out with PrPsc from brain homogenates and in intact animals. An Administrative Core, Animal Core and Tissue Core are also proposed. In addition to characterizing all tissues, the Tissue Core will continue to define classification and phenotypic spectrum of human prion diseases. This Program Project takes advantage of the close interactions and diverse expertise of several investigators to propose a multidisciplinary study of various aspects of PrPsc. As PrPsc has emergedas the central player in prion diseases, the studies proposed will lead to a clearer understanding of the pathogenesis in these diseases.